Advances in cancer therapeutics have occasioned a strategic shift away from generally cytotoxic drugs toward agents that specifically target tumor cells by modulating the pathways or processes that are inappropriately regulated in the tumor. Despite this shift in emphasis, the development of such mechanism-based therapies has been slow. We have recently developed cultured cell-based assays for signaling by the Hedgehog (Hh) family of secreted proteins that have the potential to significantly accelerate the discovery process. The Hh signaling pathway plays an important role in embryonic development, but when inappropriately activated after embryogenesis, is associated with human cancers such as basal cell carcinoma (BCC; 750,000 new cases per year in the U.S.), and medulloblastoma, the most common brain tumor in children. We propose to screen for cellular components with roles in the Hh pathway, using the complete Drosophila genome sequence as the basis for double-stranded RNA-mediated inactivation in Drosophila cells to systematically survey all genes. The availability of complete mouse and human genome sequences will make it possible to rapidly identify mammalian homologues that represent potential drug targets. Simultaneously, we will use our mammalian cell-based assays to screen chemical libraries for small molecules that modulate Sonic Hh (Shh) signaling. By mapping the action of these compounds and of mammalian components to points within the Hh pathway, we will assign potential drug leads to their cellular targets, thus facilitating further enhancement of drug potency and specificity. The value of such drug leads as potential chemotherapeutic agents is highlighted by our recent studies of cyclopamine, which demonstrate that this plant-derived compound is a specific inhibitor of Shh signaling, that adult mice tolerate prolonged treatment with this drug, and that this drug has anti-tumor activity in a mouse model of medulloblastoma. In addition to identification of new drug leads and cellular targets within the Hh pathway, our approach will serve as a general model for similar systematic approaches to anti-tumor therapies for tumors associated with the derangement of other cellular processes or signaling pathways.